Neutralization of LINGO-1 during In Vitro Differentiation of Neural Stem Cells Results in Proliferation of Immature Neurons

Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain

Cellular and Molecular Responses to Traumatic Brain Injury

Traumatic brain injury (TBI) is a relatively unknown disease considering the tens of millions of people affected around the world each year. Many TBI patients die from their injuries and survivors often suffer from life-long disabilities. The primary injury initiates a variety of cellular and molecular processes that are both beneficial and detrimental for the brain, but that are not fully understood. The focus of this thesis has been to study the role of astrocytes in clearance of dead cells after TBI and to identify injury specific proteins that may function as biomarkers, by using cell cultures, animal models and in cerebrospinal fluid (CSF) from TBI patients. The result demonstrates a new function in that astrocytes, the most numerous cell type in the brain, engulf dead cells after injury both in cell cultures and in adult mice and thereby save neurons from contact-induced apoptosis. Astrocytes are effective phagocytes, but degrade the ingested dead cells very slowly. Moreover, astrocytes express the lysosome-alkalizing proteins Rab27a and Nox2 as well as major histocompatibility complex class II, the receptors on which antigens are being presented. By lowering the pH of the lysosomes with acidic nanoparticles, the degradation increases, but the astrocytes still remained less effective than macrophages. Taken together, the data indicates that the low acidification in astrocytes can preserve antigens and that astrocytes may be able to activate T cells. The expression and secretion of injury-specific proteins was studied in a cell culture model of TBI by separate mass spectrometry analysis of cells and medium. Interestingly, close to 30 % of the injury-specific proteins in medium are linked to actin, for example ezrin of the ezrin/radixin/moesin (ERM) protein family. Ezrin, but none of the other ERM proteins or actin, is actively secreted after injury. Extracellular ezrin also increases in CSF in response to experimental TBI in rats and is present in CSF from TBI patients, indicating that ezrin is a potential biomarker for TBI.

Vardagsteknik i förskolan : hur tas tekniken tillvara?

Syftet med denna studie är att synliggöra hur teknik tas tillvara i vardagen, är det i ögonblicket eller är det i de iscensatta aktiviteterna? Enligt läroplan för förskolan Skolverket (2010) bör barnen utmanas i aktiviteter och i lek men samtidigt locka och utmanas i sitt lärande som även främjar barns lärande och utveckling inom teknik. Studien genomfördes genom kvalitativa intervjuer med tio förskollärare på sex olika förskolor i Mellansverige. Vi transkriberade intervjusvaren tillsammans, och läste sedan svaren vid flera tillfällen och kodade innehållet. Vi använde oss av olika nyckelord så som förhållningssätt, tekniska verktyg, olika åldrar, material, miljö, pedagogisk dokumentation. Vi gav de olika nyckelorden olika färg för att särskilja dem. Tillsammans sammanställde vi likheter och skillnader utifrån de svar vi fått av förskollärarna om hur de tar tillvara på vardagstekniken i förskolan. I denna studie har det framkommit att förskollärarna tycker att teknik finns överallt och i allt de gör på förskolan. De uttrycker själva att de måste bli bättre på att synliggöra den och benämna den för barnen. De efterlyser mer utbildning inom detta område Vi tror att flera av de medverkande förskollärarna har fått upp ögonen för teknik i förskolan på grund av att vi har både gjort vår pilotstudie men även denna studie inom teknik på förskolorna. Genom våra intervjufrågor tror vi att förskollärarna synliggör tekniken mer, samt reflekterar kring teknik i förskolan både med sig själv och med sina kollegor. Detta tror och hoppas vi har betydelse för deras fortsatta utveckling inom tekni

Engulfing Astrocytes Protect Neurons from Contact- Induced Apoptosis following Injury

Clearing of dead cells is a fundamental process to limit tissue damage following brain injury. Engulfment has classically been believed to be performed by professional phagocytes, but recent data show that non-professional phagocytes are highly involved in the removal of cell corpses in various situations. The role of astrocytes in cell clearance following trauma has however not been studied in detail. We have found that astrocytes actively collect and engulf whole dead cells in an in vitro model of brain injury and thereby protect healthy neurons from bystander cell death. Time-lapse experiments showed that migrating neurons that come in contact with free-floating cell corpses induced apoptosis, while neurons that migrate through groups of dead cells, garnered by astrocytes, remain unaffected. Furthermore, apoptotic cells are present within astrocytes in the mouse brain following traumatic brain injury (TBI), indicating a possible role for astrocytes in engulfment of apoptotic cells in vivo. qRT-PCR analysis showed that members of both ced pathways and Megf8 are expressed in the cell culture, indicating their possible involvement in astrocytic engulfment. Moreover, addition of dead cells had a positive effect on the protein expression of MEGF10, an ortholog to CED1, known to initiate phagocytosis by binding to phosphatidylserine. Although cultured astrocytes have an immense capacity for engulfment, seemingly without adverse effects, the ingested material is stored rather than degraded. This finding might explain the multinuclear astrocytes that ar

Bystander cell death is induced after direct contact between free-floating cell corpses and healthy, migrating neurons.

<p>(A) The cell corpse (yellow arrowhead) is initially connected to the healthy neuron's (red star) axon (0 h), but apoptosis is not induced until the two cell bodies make contact. After the cell body contacts the cell corpse (1 h 10 min, yellow arrowhead), the healthy neuron (red star) rounds up (1 h 20 min), blebs (1 h 30 min) and dies (2 h). First picture is taken 5 h and 50 minutes after injury and time indicated in the pictures is time lapsed after the first image. Scale bars = 10 µm, the dashed lines represent the scratch. (B) To analyze the frequency of bystander cell death, all neurons in 4 different time-lapse films were tracked. In total we found 26 neurons that came in contact with free-floating dead cells. Of these 26 neurons, 21 neurons (80.8%) died within the experiment. The squares represent the survival time for each of these 21 neurons after contact with the dead cell and lines represent the median±interquartile range.</p

Neurons, astrocytes and oligodendrocytes respond differently to injury <i>in vitro</i>.

<p>(A) Phase contrast micrograph of a scratch injury to a mixed cell culture of neurons, astrocytes and oligodendrocytes. Injury was induced with a scalpel and 22 hours after the cut the cell cultures were fixed in 4% PFA and stained with specific antibodies against (B) neurons (β III tubulin, red), (C) astrocytes (GFAP, green) and (D) oligodendrocytes (CNPase, red). (E–F) Many TUNEL positive cells (green) with condensed nuclei (blue) were found to be in close contact with (E) GFAP positive astrocytes, but not with (F) neurons and only occasionally with (G) oligodendrocytes. Most of the (H) nestin positive cells were associated with TUNEL positive cells. (I) Quantification of dead, TUNEL positive cells that overlapped with either cell type show that astrocytes are the primary cell to be associated with dead cells. Dashed lines represent the scratch. Scale bars equal 50 µm (A–D) and 20 µm (E–H).</p

Astrocytes are ineffective in degrading the engulfed cells.

<p>Apoptotic cells were pre-labeled with pHrodo-dye and added to (A,C) control macrophage cultures and (B,D) neuronal cell cultures. Parallel neural and macrophage cell cultures were fixed after (A–B) 5 h or (C–D) 3 d. (A) After 5 h, macrophages had already started to degrade phagocytosed cells in the lysosomes (bright red intracellular compartments). (B,D) Astrocytes did not contain red fluorescing material at any time point, indicating that the engulfed cell corpses did not fuse with lysosomes. (C) Dead cells ingested by macrophages had been degraded after 3 days and only remnant pHrodo-dye was apparent intracellularly. (D) In contrast to the macrophages, astrocytes had accumulated more intact DAPI labeled nuclei at day 3, but did not fluoresces red, indicating that the ingested cells had not fused with lysosomes at this time. (E) BrdU labeled, apoptotic cells were added to neuronal cultures and parallel cultures were fixed after 1 and 3 days (1 d respective 3 d in graph) or were carefully washed after 1 or 3 days and incubated for an additional 2 days in medium without apoptotic cells (1 d+2 d respective 3 d+2 d in graph) prior to fixation. Counting of the ingested BrdU+ nuclei show that astrocytes continued to accumulate cell corpses during the 1 day to 3 days of incubation, but after 2 days in medium without dead cells the ingested dead cells had still not been degraded. Error bars represent SEM.</p

Astrocytes engulf dead cells following neural injury.

<p>(A–B) Embryonic astrocytes have engulfed dead cells with highly condensed nuclei. Twenty-two hours after scratch injury, the cultures were fixed and astrocytes were identified with specific antibodies against GFAP. Phalloidin and DAPI labeling was used in order to visualize the actin cytoskeleton and cell nuclei, respectively. Condensed DAPI positive nuclei are present in cytoplasmic vacuoles (white arrows) within the viable astrocyte (white asterisk) and not in direct contact with the cytoskeleton of the astrocyte, indicating a macropinocytotic engulfment mechanism. (C) To prove that the cells with condensed nuclei found within astrocytes are <i>de facto</i> dead, we labeled cultures with the apoptotic marker, TUNEL. (D) TEM image of an astrocyte that appear to be in the process of engulfing a dead cell (red star). The dead cell displays the hallmark chromatin traits of apoptosis, but also secondary necrosis as seen by the ruptured cell membrane. The astrocyte contains a previously ingested dead cell (white star) in a spacious vesicle. The astrocyte is marked by an A and the nucleus is denoted Nu. (D') The area marked by a yellow rectangle in D at higher magnification. The ingested dead cell (white star) is contained in a membrane-enclosed compartment (black arrowheads) that is closely linked to the membrane on one side of the dead cell and then diverge to create the vesicle (denoted V) seen in D. Scale bars: 20 µm (A–C), 5 µm (D), 200 nm (D').</p

Molecular mechanisms behind the macropinocytosis-like engulfment in astrocytes.

<p>(A) TEM image of an astrocyte in the process of engulfing a dead cell (white star) show interaction points (black arrowheads) indicative of receptor/ligand interaction. Dead cells were added to the culture and incubated for 22 h before fixation and prepared for TEM. (B) Phagocytic genes are expressed in the neural cultures. qRT-PCR data showing the expression of <i>GFAP</i>, <i>Megf10</i>, <i>Crk</i>, <i>Rac1</i> and <i>Mfge8</i>. The average of 5 independent injured and 5 uninjured neural cultures are presented. <i>Megf10</i>, <i>Crk</i>, <i>Rac1</i> and <i>Megf8</i> were expressed in all the cultures. Error bars represent SEM. (C) The protein levels of MEGF10 is up-regulated after addition of dead cells. Fresh medium with or without dead cells were added to cell cultures, differentiated for 8 days. The cells were incubated for an additional 1 day (9 days in graph) or 3 days (12 days in graph). Cell lysates were analyzed for MEGF10 expression by Western blot and β Tubulin served as a loading control. (D–E) Engulfed cells are found in macropinocytotic-like vacuoles. Twenty-two hours after scratch injury, the cultures were fixed and astrocytes were identified with specific antibodies to GFAP. Phalloidin and DAPI labeling was used in order to visualize the actin cytoskeleton and cell nuclei, respectively. To be counted as engulfed, the dead cells (condensed nuclei) had to be situated in cytoplasmic vacuoles (white arrowheads) within the astrocytes. (F–H) The highly vacuolized astrocytes appear most active in cell corpse clearing. (F–G) The micrographs show a highly vacuolized astrocyte before engulfment (F) and approximately 28 h later the same astrocyte have ingested several dead cells (G). First picture is taken 25 minutes after injury and time indicated in the pictures is time lapsed after the first image. The dashed line represents the cut. (H) The vacuoles (black arrowheads) can be seen in transmission electron microscope as round vesicles, some apparently empty whereas others contain debris. An astrocyte-ingested dead cell is marked by a white star and the nucleus by Nu. Scale bars: 200 nm (A), 10 µm (D–G), 2 µm (H).</p

Engulfing astrocytes are present in the brain following acute injury.

<p>(A) Astrocytes derived from the adult brain engulf cell corpses. Mixed cell cultures derived from SVZs of adult animals were injured and fixed after 22 hours. Dead cells were identified by TUNEL staining (green), astrocytes with specific antibodies against GFAP (red) and nuclei by DAPI (blue). Confocal micrograph shows a dead, TUNEL positive cell within an astrocyte (white arrows). (B–M) Traumatic brain injury in mice elicits astrocytic engulfment of dead cells at the site of injury. Animals that received CCI-injury were perfused after one (<i>n</i> = 5), three (<i>n</i> = 5) or seven (<i>n</i> = 5) days and cryostat brain sections were labeled with TUNEL (red), DAPI (white) and antibodies against GFAP (green). Confocal micrographs show dead cells within the astrocytes at (B–E) one, (F–I) three and (J–M) seven days post-injury. (N) Scatter plot of the percent TUNEL+ cells associated with viable astrocytes in each animal 7 days post-CCI. For each five animals, two separate sections from bregma levels −1.0 and −2.0 mm, were counted and the percentage in 8–10 fields for each level (40× magnification) were plotted. Scale bars = 5 µm.</p